The shortage of cells exhibiting glucose-responsive insulin secretion has been a major obstacle to the development of widely applicable cell transplantation therapies for diabetes. To address this problem, the ultimate goal of the proposed studies is to develop cell lines from human beta-cells. This would provide a large quantity of cells, grown in vitro, that retain glucose-responsive insulin secretion or the ability to differentiate and acquire this property. Such cells could then be used for cell transplantation therapies for diabetes. Human beta-cell lines are produced by expressing growth stimulatory genes in the cells, resulting in extracellular matrix and growth factor independent growth in vitro. A human beta-cell line, betalox5, can be induced to exhibit glucose-responsive insulin secretion in vitro and in vivo following transplantation into nude mice. These results represent the achievement of a major milestone towards developing a cell transplantation therapy for diabetes. The focus of this proposal is on developing our human beta-cell line technology to the point where large animal and human clinical trials are feasible. Specifically, improved cell lines expressing alternative combinations of growth stimulatory genes will be developed. Improved methods for inducibly deleting the growth stimulatory genes using the cre-lox recombinase system will be studied.